PROJECT SUMMARY Endometrial cancer (EC) is the most common gynecologic malignancy in the US. Most cases are sporadic. However, 4-5% of women with EC develop their disease because they carry inherited DNA mismatch repair (MMR) gene mutations and, as such, have Lynch syndrome (LS). An additional 25% of ECs have acquired defective DNA MMR, typically associated with epigenetic silencing of the MLH1 repair gene. Identifying LS in EC patients provides opportunities for cancer prevention for the patient and her family members, and because of this, tumor-based screening for LS in EC has been recommended. Recognizing MMR deficiencies and other genetic abnormalities in ECs is also important in treatment planning and evaluation of new therapies. At present, LS screening relies on a series of IHC and DNA tests that guide genetic counseling and testing for germline mutations. Population-based germline mutation testing is, however, time-prohibitive because of the need for pre-test counseling for all subjects, with only ~1/20-1/25 having LS, and the potential for incidental findings for other cancer susceptibility loci. We propose development of robust, low-cost methods for detection of LS mutations using tumor DNAs that can be used for all EC patients. Our testing approach will ensure that more EC patients with LS, and their family members with LS mutations, can benefit from intensified cancer surveillance. Furthermore, the tumor analysis methods proposed include tests that allow for classification of molecular EC subtypes that can direct selection of adjuvant therapies. Our experimental design is powered by unprecedented biospecimen resources linked to clinicopathologic data. Our analyses are focused on use of specimens prepared in CAP-approved laboratories, paving the way for rapid translation to clinical test development. Three specific aims are proposed to allow us to develop clinically relevant tumor-based testing for inherited cancer susceptibility and profiling for molecular classifications that will inform adjuvant treatment decision making and further translational research. Aim 1: To further develop robust methods for tumor-based detection of germline LS mutations, MSI and copy number alterations. Aim 2: To determine the efficacy of screening for Lynch syndrome among all newly diagnosed endometrial cancer patients using upfront next-generation sequencing of mismatch repair genes as compared to standard of care sequential testing. Aim 3: To determine the prognostic significance of a clinically-applicable molecular classification system for endometrial cancers.